Traditional methods for recombining and cloning DNA involve using restriction enzymes and DNA ligase to cut and create cohesive or blunt ends in DNA and then to recombine and link the DNA. Restriction enzymes facilitate the preparation of the desired DNA segments and the process of recombining DNA (Smith et al., 1970, J. Mol. Biol. 51(2):379-391). DNA ligase links two segments of DNA, an insert and a vector such as a plasmid (Weiss et al., 1968, J. Biol. Chem. 243(17):4543-4555). The linked or ligated DNA can be introduced into a microorganism such as E. coli for cloning (Cohen et al., 1973, Proc. Natl. Acad. Sci. USA 70(11):3204-3244). Restriction enzymes aid in recombination by creating cohesive ends on DNA fragments which are used for properly aligning DNA fragments. When the DNA has two different cohesive ends after restriction enzyme treatment, the orientation of the insert in relation to the vector can be controlled.
One common problem with the traditional recombination and cloning method is that nucleic acids do not always have suitable restriction sites. When joining protein coding sequences, restriction sites must flank the sequences of interest and possess cohesive ends compatible with the sites in the vector or another coding sequence into which it is to be inserted. Continuity of the reading frames must be preserved after ligation so that the correct protein results from subsequent DNA transcription and RNA translation processes.
In practice, naturally occurring restriction sites that satisfy such requirements are often not present. A known method of preserving the reading frame is to create new restriction sites using oligonucleotides as linkers or adapters or polymerase chain reaction (PCR), a step which requires one or more rounds of cloning. New restriction sites can be created in PCR by including one or more restriction sites in the PCR primers that are synthesized by chemical means. However, the choice of a new restriction site is often limited because identical sites may be present within the cloning vector or in the sequence to be cloned.
The traditional method is complicated and time consuming, as is evident from a consideration of these problems in the art.
A method was recently described which can be independent of natural restriction sites or in vitro ligation (Ma et al., 1989, Gene 58:201-216; Oldenburg et al., 1997, Nucleic Acids Research 25:451-452). This method is an in vivo method for plasmid construction that takes advantage of the double-stranded break repair pathway in a yeast to achieve precision joining of DNA fragments. However, this method requires synthesis of linkers (60-140 base pairs) from short oligonucleotides and requires assembly by enzymatic methods into the linkers needed (Raymond et al., 1999, BioTechniques 26(1):134-141).
The invention described herein overcomes the problems inherent in the traditional cloning methods and does not require use of restriction endonucleases or DNA ligase.